This invention relates to characterization of particles suspended in a fluidic medium. In particular, the invention is directed to an apparatus and method for simultaneously characterizing the fluorescence and motility of cell, bacteria and particles in fluids.
Conventional cell characterization systems generally employ characterization techniques that are directed to either conventional fluorescence measurements or conventional motility measurements using primarily visible light. Cell characterization systems utilizing visible light to interrogate the specimen provide standard motility parameters and cell count. Systems employing a fluorescence technique provide standard fluorescent parameters such as membrane integrity.
The motion of unstained cells is utilized to identify motion characteristics of living cells in standard microscopy. In the context of semen analysis identifying the motion characteristics includes determining for example the sperm motility and mean sperm velocity where sperm motility is understood as that fraction of sperm moving among the sperm in the specimen sample. Cell characterization, and primarily sperm motility characterization, is regularly utilized for animals, such as horses and bulls, to establish the reproductive quality of their sperm which is integral in the evaluation of their breeding potential. In addition, motility analysis is important in the diagnosis of reproductive abnormalities in human males.
Conventional techniques of cell characterization to measure standard motility parameters primarily utilize illumination in the visible light spectrum. A radiation generating source, emitting radiation in the visible light spectrum, is directed onto a specimen and the light is refracted by both the fluidic medium and the cells contained therein. The refracted light is conditioned and directed, by appropriate optics, onto a light sensitive device which measures light incident thereon. Utilizing conventional electronics and data processing techniques, the refracted light is analyzed for desired information, for example, standard cell motility parameters.
Examination of fluorescing cells is utilized in microbiology to provide information on cell membrane integrity and, with respect to sperm cells, acrosomal integrity. The acrosome, a baglike structure surrounding the head of the sperm cell, must be substantially intact and able to withstand acrosomal reaction for spermatozoa to penetrate the zona pellucida surrounding the ovum. The characterization of the acrosome integrity is of great importance in reproduction study and abnormality diagnosis. In addition to acrosomal integrity, characterizing fluorescing cells provides detailed cell fluorescence and motility information, for example, cell brightness and cell velocity.
Generally, fluorescence is induced by the illumination of a specimen stained with an appropriate fluorophore wherein the wavelength of the illumination is substantially within the fluorophore absorption peak bandwidth. The fluorophore absorbs the shorter wavelength radiation and, due to the fluorescing characteristics of the fluorophore, causes photon emission at a wavelength longer than that of the irradiating illumination. In addition, the emitted photons tend to have a wavelength within the visible light wavelength spectrum. The wavelength of the emitted photons is substantially dependent upon the fluorescence characteristics of the fluorophore and the wavelength of the irradiating illumination.
Fluorescent light characterization systems frequently employ ultra violet light to stimulate cells which are stained with a fluorophore dye. The ultra violet light generating source is directed onto a specimen stained with a fluorescence dye and, as described above, the specimen absorbs the ultra violet radiation. The dye, within both the cells and fluidic medium comprising the specimen, emits photons having wavelengths in the visible light spectrum. As in the case of refracted light, the emitted light is conditioned and directed, by appropriate optics, onto a light sensitive device which measures light incident thereon. Generally, the radiation sensitive device is an eyepiece or a camera in an instrument which often is a microscope. Appropriate electronics and data processing techniques are again used to obtain desired information.
A primary shortcoming of the conventional fluorescent light characterization systems is the techniques employed to induce fluorescence. The excitation radiation is generally a continuously irradiating source. The intense irradiation of the specimen is phototoxic and consequently destroys the living cells under investigation. In addition, these systems employ excitation radiation having wavelength characteristics that are also substantially phototoxic and, as described above, generally results in destruction of the living cell. As a result of the phototoxic effects, systems employing such techniques generally fail to accurately characterize general cell motility parameters as well as membrane integrity and cell fluorescent intensity.
In addition, conventional fluorescent light characterization system do not employ techniques to permit rapid assessment of the quantitative estimates of fluorescent brightness directly. This feature would permit a determination of the quantity of fluorophore present in the cell on an absolute scale. Conventional systems utilize a technique providing relative estimates of the amount of fluorophore present and thus fail to accurately characterize a crucial property of the cell.